By the development of recombinant DNA technique in recent years, mass production of heterogeneous proteins has become possible using microorganisms, particularly Escherichia coli.
Improvement of the frequency of expression of heterogeneous genes in host cells is an important subject.
Specifically, in order to elevate transcription efficiency, as the promoter which is genetic information necessary for the initiation of transcription, lactose (lac) promoter (referred to as lac promoter hereinafter), tryptophan (trp) promoter (referred to as trp promoter hereinafter) and the like have been employed. Further, a hybrid promoter (tac promoter) of trp promoter and lac promoter has been designed. They are employed for expression of heterogeneous genes [K. Itakura, et al.: Science 198, 1056-1063(1977), D. V. Goeddel, et al.: Nature 287, 411-416 (1980), H. A. deBoer, et al.: Proc. Natl. Acad. Sci. USA, 80, 21-25 (1983)].
The region which RNA polymerase recognizes and binds to initiate transcription is named "promoter". It is well known that transcription efficiency depends on the DNA base sequence of the "promoter". According to the conventional numbering system of DNA base sequences, the promoter of Escherichia coli comprises the region around "-35", which is usually named "-35" region, the region around "-10", which is named "-10" region or Pribnow box, and the region of "+1", i.e. transcription initiation site which is a purine base such as adenine and guanine in most promoters. "-35" region is defined as a recognition site of RNA polymerase. According to the statistical analysis of base sequences of various Escherichia coli promoters, it is clarified that the consensus sequence is TGTTGACANTTT wherein A is adenine, T is thymine, G is guanine, C is cytosine and N is any of A, T,G and C. The most frequently appearing part of the consensus sequence is TTGACA. On the other hand, "-10" region is recognized as an RNA polymerase binding site and the consensus sequence thereof is TATAATG [M. Rosenberg & D. Court: Ann. Rev. Genet. 13. 319 (1979)].
As the promoters generally used to express a gene of eukaryotes, lac UV5 promoter [F. Fuller: Gene 19, 43 (1982)], trp promoter [G. N. Bennet, et al.: J. Mol. Biol. 121, 113 (1978): D. V. Goeddel, et al.: Nature 287, 411 (1980)] are known. In the case of lac UV5 promoter, the "-10" region (TATAATG) is completely identical with the consensus sequence of "-10" region of Escherichia coli promoter described above but the "-35" region (GCTTTACACTTT) is not identical with the consensus sequence. Reversely, in the case of trp promoter, the "-35" region (TGTTGACAATTA) is almost identical with the consensus sequence but the "-10" region (TTAACTA) is not identical with the consensus sequence.
H. A. deBoer, et al. of Genentech Inc. constructed a hybrid promoter by ligating a DNA fragment containing "-35" region of trp promoter and the 5' flanking region upstream therefrom and a DNA fragment containing "-10" region of lac promoter and lac operator, utilizing superiority of trp promoter and lac UV5 promoter. The hybrid promoter was named tac promoter and has been employed to express human growth hormone. The tac promoter is characterized in that "-35" region derived from trp promoter and "-10" region derived from lac promoter are constructed so as to function as a promoter and both regions are almost identical with the consensus sequences [H. A. deBoer, et al.: Proc. Natl. Acad. Sci. USA 80, 21 (1983)].
The thus constructed tac promoter was proved to have transcription activity which is about 3.5 times stronger than that of trp promoter and 2-3 times stronger than that of lac promoter. Further, H. A. deBoer, et al. constructed rac promoter which is a hybrid promoter of the promoter of ribosome RNA operon known as an extremely strong promoter and lac promoter to use for expression of human growth hormone (Japanese Published Unexamined Patent Application No. 194790/82).
Thus H. A. deBoer, et al. provided a method of constructing novel hybrid promoters by religating selectively and functionally the DNA fragment containing 5' flanking region and "-35" region and the DNA fragment containing "-10" region utilizing superiority of the two promoters. The method may be useful for the construction of hybrid promoters which have a strong transcription activity and an ability to control expression. However, the method is not adequate to construct promoters which can maximally exhibit the transcription ability of Escherichia coli. The present inventors have studied to elevate the transcription activity of trp promoter and tac promoter.
H. A. deBoer et al. reported the following factors affecting the strength of promoter [H. A. deBoer, et al.: "Promoters: structure and function" R. L. Rodriguez, et al. M. J. Chamberlin ed. (Praeger Co.) 462-481 (1982)]:
(1) Base sequence of "-10" region PA0 (2) Base sequence of "-35" region PA0 (3) Distance between "-10" and "-35" regions PA0 (4) AT content in 5' flanking region upstream from "-35" region PA0 (5) Appropriate combination of these factors
Factors (1),(2) and (3) are known to have an important effect on the strength of a promoter. The present inventors presumed that the factor (4) would have an important effect on the strength of a promoter.
Some strong Escherichia coli promoters are known to be abundant with adenine (A) and thymine (T) in the 5' flanking region and to contain a part (AT) wherein A and T are linked successively. [Z. Humayun, et al.: J. Mol. Biol. 112, 265 (1977), G. T. Horn, et al.: J. Biol. Chem. 256, 1998 (1981), G. N. Bennet, et al.: J. Mol. Biol. 121, 113 (1978), K. Nakamura, et al.: Cell 18, 1109 (1979)].
Further, it is known that the AT content in the 5' flanking region affects the strength of a promoter. That is, G. T. Horn, et al. reported that the strength of P.sub.L promoter of .lambda. phage was reduced by the deletion of the 5' flanking region abundant with AT [G. T. Horn, et al.: J. Biol. Chem. 256, 2003 (1981)]. Further, R. D. Klein, et al. reported that the in vitro transcription activity of lac promoter was enhanced by inserting a double stranded DNA poly(A).multidot.poly(T) of about 70 bp into the site upstream from the "-35" region of lac promoter [J. Biol. Chem. 257, 12954 (1982)]. Therefore, it is presumable that endowment of a sequence abundant with AT to 5' flanking region increases the strength of a promoter.
The 5' flanking region of P.sub.L promoter has the following characteristics (refer to Table 1). First of all, two parts abundant with AT are included in the 5' flanking region. The two parts abundant with AT are -186 to -155 and -101 to -75 according to the conventional numbering wherein the transcription initiation site of P.sub.L promoter is indicated by +1.
TABLE 1 __________________________________________________________________________ DNA base sequence of P.sub.L promoter __________________________________________________________________________ ##STR1## ##STR2## ##STR3## ##STR4## __________________________________________________________________________
G. T. Horn, et al. reported that the deletion of both AT-abundant parts resulted in reduction of the strength of P.sub.L promoter but did not change the function of P.sub.L promoter [G. T. Horn, et al.: J. Biol Chem., 256, 2003 (1981)]. A "-35"-like base sequence present in the part from -60 to -49 also may have relation to the strength of the promoter. In the case of the base sequence of P.sub.L promoter, the sequences of "-35" region and "-10" region are very similar to but not identical with the respective consensus sequences. The similarity of the sequences to the consensus sequences is in the same degree with that in the case of trp promoter. However, P.sub.L promoter has a stronger transcription activity than trp promoter. This may be caused by the difference of the base sequences in the 5' flanking regions. Therefore, a promoter wherein the 5' flanking region is replaced with that of P.sub. L promoter may be strengthened.
In order to replace 5' flanking region, the method developed by H. A. deBoer, et al., that is, the method which comprises recombining "-35" region containing 5' flanking region and "-10" region to construct a new hybrid promoter (Japanese Published Unexamined Patent Application No. 194790/82) is applicable. However, the method has a possibility to impair the function inherited by a promoter since the recombination of "-35" region and "-10" region is variable. For example, when the method is applied to very excellent tac promoter to construct a stronger promoter by replacing the 5' flanking region with another one, the 5' flanking region and the "-35" region are liable to be simultaneously replaced with other DNA fragments, whereby the "-35" region inherent in tac promoter is lost.
The present inventors have found a process for replacing only the 5' flanking region as described hereinafter, that is, a process for replacing only the 5' flanking region utilizing a restriction site which is present around or upstream from the "-35" region.